An essential function of sphingolipids in yeast cell division.

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Ceramides are essential bioactive lipids and sphingolipid precursors involved in numerous eukaryotic physiological processes.
• Ceramide synthases, a family of enzymes, dictate ceramide structure based on fatty acyl CoA substrate specificity.
• High ceramide concentrations are generally considered toxic to cells.

Purpose of the Study:

• To characterize a putative ceramide synthase from cotton for its enzymatic activity and substrate specificity.
• To investigate the physiological consequences of producing specific ceramide chain lengths in yeast.
• To explore the essential roles of sphingolipids in Saccharomyces cerevisiae by enabling AUR1 gene deletion.

Main Methods:

• Genetic engineering of Saccharomyces cerevisiae to replace endogenous ceramide synthase with a cotton-derived enzyme.
• Analysis of ceramide production and chain length in the engineered yeast strain.
• Phenotypic analysis of yeast strains with AUR1 deletion and altered ceramide synthesis, focusing on cell division.

Main Results:

• The cotton enzyme functions as a ceramide synthase, specifically producing C18 ceramides in yeast, unlike the native C26 ceramides.
• Accumulation of C18 ceramides in yeast is non-toxic, enabling the deletion of the essential AUR1 gene.
• Deletion of AUR1 in this engineered strain leads to delayed cytokinesis and incomplete cell separation, despite normal nuclear division.

Conclusions:

• The cotton protein is confirmed as a functional ceramide synthase with a preference for C18 acyl CoA.
• The non-toxicity of C18 ceramides in yeast provides a novel tool for studying sphingolipid metabolism and essential gene functions.
• Sphingolipids play a critical role in regulating the timing and completion of cytokinesis in Saccharomyces cerevisiae.